Phosphatidylinositol mannosides (PIM) and their multiglycosylated counterparts, lipomannan (LM) and lipoarabinomannan (LAM) are complex glycolipids/ lipoglycans that are found in the envelopes of all mycobacterial species. They are important immunomodulatory molecules in the course of tuberculosis and leprosy as well as key ligands promoting the entry of mycobacteria into phagocytic and non-phagocytic cells. Although much progress has been made over the last 15 years in deciphering the structures and structure-function relationships of these molecules, little is known about their biogenesis. The elucidation of their biosynthetic pathways, in addition to providing fundamental knowledge about the biochemistry of Mycobacterium tuberculosis, would lead to the discovery of essential enzymes that could represent crucial targets for novel anti-tuberculosis drugs. The availability of defined M. tuberculosis and M. bovis BCG mutants deficient in some aspects of PIM/ LM and LAM synthesis would also enable a precise measurement of the contribution of these molecules to the immunopathogenesis of tuberculosis and suggest new vaccine strategies against this disease. We propose to exploit the recent knowledge of the structures of LAM-like molecules produced by actinomycetes together with the availability of a growing number of genome sequences of lipoglycan-producing actinomycetes to perform genome comparison studies, identify glycosyltransferases involved in the biogenesis of PIM, LM and LAM and construct lipoglycan-deficient mutants of mycobacteria. Using the CS- 35 anti-LAM monoclonal antibody and other antibodies that we have developed, we also propose to screen transposon mutant libraries of M. tuberculosis, M. bovis BCG, and M. smegmatis for mutants defective in some aspects of LAM synthesis. Mutants will be biochemically characterized using conventional methods and novel analytical tools, cell-free assays will be developed for the newly identified biosynthetic enzymes and the three-dimensional structure of these proteins will be determined. Finally, the interactions of PIM/ LM/ LAM mutants with host cells and their immunomodulatory properties will be investigated.